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Risk Assessment Summary Conducted Pursuant to the New Substances Notification Regulations of the Canadian Environmental Protection Act, 1999

Trichoderma reesei P59G NSN 11909

Table of Contents

This document has been prepared to explain the regulatory decision taken under Part 6 of the Canadian Environmental Protection Act, 1999 (CEPA 1999) regarding the manufacture or import of Trichoderma reesei P59G by Iogen Corporation in a contained facility located in Ottawa.

The New Substances Branch of Environment Canada and the New Substances Assessment and Control Bureau of Health Canada have assessed the information submitted by Iogen Corporation and other available scientific information in order to determine whether T. reesei P59G is toxic[1] or capable of becoming toxic as described in section 64 of CEPA 1999.

Regulatory Decision

Based on the hazard and exposure considerations, the joint risk assessment conducted by Environment Canada and Health Canada concluded that Trichoderma reesei P59G is not considered to be toxic to the Canadian environment or human health as described in section 64 of the CEPA 1999.

Therefore, the manufacture in or import to a contained facility of T. reesei P59G for use in the contained facility or for export only, may proceed after November 9, 2002.

This evaluation does not include an assessment of human health risk in the occupational environment nor does it include an assessment of the potential exposure and risk to humans associated with the use of the organism in or as an item that falls under the purview of the Food and Drugs Act.

NSN Schedule: XVI (manufacture or import to a contained facility of a micro-organism that is not for introduction outside a contained facility or is for export only)[2]Organism Identity:Trichoderma reesei P59G Notifier: Iogen Corporation, 310 Hunt Club Rd. East Ottawa, Ontario K1V 1C1 CanadaDate of decision: November 9, 2002 Proposed use: Commercial production, in a contained facility, of a β-glucosidase enzyme by genetically engineered Trichoderma reesei P59G.

Strain History/Genetic Modification

Trichoderma reesei P59G was derived from an auxotrophic mutant of the parental strain BTR213 through the introduction of the linear transformation vector pc/x bg-pyr4-TV. The selection cassette used in the construction of the transformation vector contains a Neurospora crassa gene functioning as a selectable marker. The expression cassette consisted of the native T. reesei b-glucosidase structural gene (bgl1) under the control of T. reesei regulatory sequences. Parental strain BTR213 was obtained from T. reesei M2C38 (ATCC 74252) using classical mutagenesis techniques. Strain M2C38 is a derivative of the T. reesei strain RUTC30 (ATCC 56765), a mutagenic derivative of the founder strain QM6a (ATCC 13631) which was isolated in the Solomon Islands from cotton canvas during World War II (Kuhls et al., 1996).

Hazard considerations

In addition to the information provided by the notifier, a review of in-house reference material and a comprehensive search of the scientific literature were conducted to gather information on potential harmful environmental and human health effects attributable to T. reesei.

Trichoderma species are common soil saprophytes and are metabolically versatile, aerobic, mesophilic, imperfect fungi (Nevalainen et al., 1994). The Trichoderma species are differentiated primarily by patterns of conidiophore branching and conidia morphology. They are widespread in nature, quick-growing, easy to culture and they can produce large amounts of conidia with long lifetime (Manczinger et al., 2002).

In general, large scale industrial manufacture of T. reesei enzyme preparations have a history of safe use in many industries including starch and animal feed processing, grain alcohol fermentation, malting and brewing, extraction of fruit and vegetable juices, pulp and paper, and textiles (Hjortkjaer et al., 1986). Based on the criteria outlined in the Organisation for Economic Co-operation and Development guidelines entitled Recombinant DNA Safety Considerations (OECD, 1986) and the European Communities Council Directive 90/219/EEC on the contained use of genetically modified micro-organisms (ECC, 1990), Trichoderma species can be regarded as safe host organisms.

Trichoderma reesei has been shown to be non-pathogenic and non-toxic to healthy laboratory animals (Hjortkjaer et al., 1986). Trichoderma reesei is not reported to be a frank pathogen of plants or animals including humans. However, this species can act as an opportunistic pathogen to immunodepressed animals under extreme experimental conditions (Hjortkjaer et al., 1986). Some Trichoderma species have been cited as rare and newly emerging fungal pathogens (Fleming et al., 2002).

While certain species of the genus Trichoderma can be used as biocontrol agents in agriculture for their ability to produce antifungal compounds against several plant pathogenic fungi, T. reesei P59G is not one of them. Some species of Trichoderma may also produce toxins under certain conditions; however, experience with T. reesei indicates that it is not likely to be toxigenic (Hjortkjaer et al., 1986). Tests conducted on commercial enzyme preparations confirm that neither antibiotics nor inhibitory substances are produced during the growth of industrial T. reesei strains (Hjortkjaer et al., 1986). A carbohydrase enzyme product manufactured by the notifier using the parental strain M2C38 was tested for aflatoxin with negative results.

Toxicity studies on native xylanasesfrom Aspergillus and Thermomyces administered orally to rats and mice did not result in adverse effects (Pederson and Broadmeadow, 2000). Native xylanases were not found to be mutagenic in the Salmonella typhimurium reverse mutation assay, nor did they cause chromosomal aberrations in cultured human lymphocytes (Pederson and Broadmeadow, 2000).

Neurospora crassa, the fungal source of the selectable marker gene used in the construction of the transformation vector, is not reported to be a frank pathogen. The N. crassa gene product makes selection of T. reesei strain P59G, from a mixture of other microorganisms, easier, and is unlikely to pose a risk to the environment since it has many functional equivalents in most living organisms.

Both T. reesei and N. crassa are listed as ‘Biosafety Level 1’ organisms by the American Type Culture Collection (ATCC). In addition T. reesei has been designated as a ‘Risk Group 1’ organism by the Office of Laboratory Security of the Public Health Agency of Canada.

The DNA fragments used in the construction of the transformation vector are well characterized and do not contain any large undefined fragments. It is unknown whether the ampicillin resistance gene present on the transformation vector was integrated onto the host genome. Nevertheless, the ampicillin resistance gene used in the construction of the transformation vector is under the control of a bacterial promoter that will not function in T. reesei. In addition, the vector DNA has been shown to be stably integrated into the chromosome without loss or rearrangement of the sequence even after several generations on non-selective media. Therefore, the potential for lateral gene transfer from this organism to humans, animals or other microbes in the environment is extremely low.

Genetic modifications performed to develop T. reesei P59G do not give rise to concerns of altered virulence or pathogenicity to humans, animals, plants or altered hazards to the environment. The phenotype resulting from the modification is well characterized and is not likely to influence the normal behavior of T. reesei.

The genetic modification resulted in a 0.42% modification of the β-glucosidase amino acid sequence and a 0.47% molecular weight increase, which is considered substantially equivalent to the native β-glucosidase enzyme from T. reesei strain M2C38. This addition to the β-glucosidase did not alter the enzymatic activity or the isoelectric point of the enzyme.

In industrial settings, allergic reactions to enzymes like β-glucosidase and other carbohydrases have been previously reported. In the present case, the use of appropriate personal protective equipment, such as personal ventilators, appears to largely eliminate this problem.

Exposure considerations

Trichoderma species, including T. reesei are common soil saprophytic fungal species found in all climate zones and are particularly prevalent in the litter of humid, mixed hardwood forests (Nevalainen et al., 1994).

Trichoderma reesei P59G is manufactured solely as an intermediate in the production of a β-glucosidase enzyme in a contained facility. The notifier indicated that the manufacturing process meets the standards for the Good Large Scale Practice (GLSP) level as defined in Appendix K of the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH, 2002). The notified strain is not intended for release outside the contained facility. Consequently, the potential exposure to the general population and the environment is expected to be low.

The notifier describes procedures which will limit potential worker exposure. These include the use of protective equipment, such as the National Institute for Occupational Safety and Health (NIOSH) approved respiratory masks with particulate filters, face shield, or safety goggles with side shields, rubber gloves, lab coats or overalls for workers who are chronically exposed to enzyme dusts or aerosols during such procedures as transfers of fermentation broths.

Precautions are in place and used by the notifier to ensure that exhaust and aerosols from the fermentor are decontaminated by UV irradiation to kill any organisms and volatiles and odours are removed by a cyclone and scrubber system. The fermentor is equipped with an alarm to indicate high pressure, foam-over and low-level and is diked in case of massive leakage. Trichoderma reesei P59G is not intrinsically hazardous, thus, inadvertent release from the manufacturing facility is not expected to pose significant risk to the environment and human health.

When enzyme production is complete, the spent cell mass is chemically inactivated using a quaternary ammonium compound with 99.999% effectiveness prior to disposal in a registered landfill or composting sites in accordance with provincial regulations. Given that T. reesei P59G lacks pathogenicity and toxicity potential, the likelihood of significant harm to the environment or human health resulting from the disposal route of exposure is expected to be minimal.

Footnotes

[1] In accordance with section 64 of the CEPA 1999, a substance is toxic if it is entering or may enter the environment in a quantity or concentration or under conditions that (a) have or may have an immediate or long-term effect on the environment or its biological diversity; (b) constitute or may constitute a danger to the environment on which life depends; or (c) constitute or may constitute a danger in Canada to human life or health.[2] Provisions relating to organisms previously contained in Part II.1 of the NSNR are now contained within the NSNR (Organisms). These came into force on October 31, 2005, and included changes to Schedule numbering. Under the NSNR (Organisms), Schedule XVI is now referred to as Schedule 2.